Sorbitol (D-glucitol) is an acyclic polyol found in a number of plant species (Kuo et al. (1990) Plant Physiol 93:151.4–1520). Sorbitol is the primary photosynthetic product in rosaceous fruits and can account for a major portion of the carbon transported from the leaf. In corn sorbitol is found in seed and silk but not in pollen and leaf and low amounts of sorbitol are detectable in developing corn kernels. Sorbitol is found in soybeans and it is suggested that the accumulation of sorbitol may play a role in facilitating hexose metabolism in germinating seedlings (Kuo et al. (1990) Plant Physiol 93:1514–1520). During germination, soybeans convert oil and soluble oligosaccharides into sucrose which is in turn converted to glucose and fructose to fuel rapid growth. Some investigators have speculated that since plant fructokinases exhibit strong substrate inhibition by fructose, the presence of a sorbitol pathway may provide a mechanism to bypass this inhibition by converting excess fructose into sorbitol. This would help facilitate the metabolism of free glucose and fructose.
The metabolism of sorbitol has been extensively studied in several plant species (Kuo et al. (1990) Plant Physiol 93:1514–1520, Loescher (1987) Physiol. Plantarum 70:553–557, Loescher et al. in: Photoassimilate Distribution in Plants and Crops, ed. Zamski et al. Marcel Dekker, Inc., New York). There are several enzyme activities involved in sorbitol metabolism. Three of these enzymes are aldehyde reductase (NADPH-dependent aldose 6-phosphate reductase), sorbitol dehydrogenase, and NADP-dependent D-sorbitol-6-phosphate dehydrogenase. Aldehyde reductase appears responsible for the conversion of glucose to sorbitol; NADP-dependent D-sorbitol-6-phosphate dehydrogenase is also involved in sorbitol synthesis, and sorbitol dehydrogenase is involved in the conversion of sorbitol to fructose. Accordingly, the availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate studies to better understand carbohydrate metabolism and function in plants, provide genetic tools for the manipulation of the sorbitol biosynthetic pathway, and provide a means to control carbon partitioning in plant cells. fragment encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a sorbitol biosynthetic enzyme selected from the group consisting of aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase.
In another embodiment, the instant invention relates to a chimeric gene encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase.